PROJECT SUMMARY AMPs are one of the components of the innate immunity of multicellular organisms, including humans. They exhibit a broad range of antibiotic activities despite their simple structures. AMPs hold promise to combat resistant bacteria due to their distinct mechanism of activity. However, AMPs' success in medicine relies on designs that are highly toxic for bacteria, yet selective enough not to harm human cells. The dif?culty is that we lack a clear picture about the dynamics of AMPs' activity that can inspire new designs. The main objective of this research proposal is to gain a precise and quantitative insight into the action of antimicrobial peptides against bacteria, at both the population level and the single-cell level. In this project, we ?rst aim to (1) quantify the population dynamics of antimicrobial peptides against bacteria. Through a series of microdilution experiments we have portrayed the dependency of the mini- mum inhibitory concentration (MIC) of AMPs on the density of bacterial cells. We propose a model based on the retention of AMPs in dead cells, which sequesters AMPs' concentration in the culture. This results in the dependency of the MIC on cell density even in very dilute cultures. We are using ?uorescent mi- croscopy on dye-tagged AMPs to support this hypothesis. The next aim of this proposal is to (2) quantify the heterogeneous action of AMPs at the single-cell level. While a bacterial population's response to AMPs is ?deterministic,? the responses of individual cells can be ?stochastic,? which carries a large level of noise and ?uctuations. We utilize a single-cell platform to perform video microscopy on cells growing in a tightly controlled environment. We hypothesize that the outcome of AMPs' treatment is binary at the single-cell level, resulting in either complete growth inhibition or normal growth. Our single-cell ex- periments can reveal these growth heterogeneities as well as the timeframe and temporal dynamics of AMP-induced cell death. Next, we plan to (3) develop a computational tool to test models for the activity of AMPs. Using quantitative data obtained from our experiments, we plan to develop a com- putational ?agent-based? simulation software for testing various hypotheses on the AMPs' action. The objective of this computational tool is twofold. First, we want to reproduce experimental observations. Second, we want to utilize the software to provide experimentally testable predictions, which can guide us towards future experimental plans. The outcome of the proposed research ?rst illuminates the population dynamics of AMPs' action ? that is, how the concentrations of AMPs and cell densities are related in determining the therapeutic results. Second, the research will uncover the heterogeneity and time scale of AMPs' actions at the single-cell level, where we can quantify the rate of death for individual cells. Both of these perspectives are of fundamental importance in terms of understanding the action and design of AMPs as therapeutic agents.